Blood coagulation results from the production of thrombin, a proteolytic enzyme inducing platelet aggregation and cleaving fihrinogen to fibrin, which stabilizes the platelet plug. A number of proenzymes and procofactors circulating in the blood interact in this process through several stages during which they are sequentially or simultaneously converted to the activated form, ultimately resulting in the activation of prothromhin to thromhin by activated Factor X (fXa) in the presence of Factor Va, ionic calcium, and platelets.
Factor X can he activated by either of two pathways, termed the extrinsic and intrinsic pathways. The intrinsic pathway, or surface-mediated activation pathway, consists of a series of reactions where a protein precursor is cleaved to form an active protease, beginning with activation of Factor XII to Factor XIIa, which converts Factor XI to Factor XIa, which, in the presence of calcium, converts Factor IX to Factor IXa. Factors IX and X can also he activated via the extrinsic pathway by tissue factor (TF) in combination with activated Factor VII (Factor VIIa; FVIIa). Activated Factor IX, in the presence of calcium, phospholipid (platelets), and Factor VIIIa, activates Factor X to Factor Xa.
Physiologically, the major pathway involved in coagulation is believed to be the extrinsic pathway, an essential step of which is tissue factor-mediated activation of Factor VII to Factor VIIa. Tissue factor is an integral membrane glycoprotein having a protein and a phospholipid component. It has been isolated from a variety of tissues and species and reported to have a molecular mass of between 42,000 and 53,000. DNA encoding tissue factor and methods for expression of the protein have now been reported, for example, in European Patent Application 0 278 776 by Genentech, Inc. and by J. H. Morrissey, et al. Cell 50:129-135 (1987).
The complex of Factor VIIa and its essential cofactor, TF, is the most potent known trigger of the clotting cascade. Factor VII is present in plasma at a concentration of 0.5 .mu.g/ml plasma. In contrast, Factor VIIa is present in plasma at trace levels of roughly 1 ng/ml. Accordingly, Factor VII is normally in considerable excess over Factor VIIa. Factor VIIa circulates with a relatively long half-life of about two hours in plasma. This is an unusual property among activated coagulation enzymes, which typically are inactivated very rapidly by various protease inhibitors in plasma.
Hemophilia A is characterized by the absence of active coagulation Factor VIII or the presence of inhibitors to Factor VIII. Hemophilia B is characterized by the absence of active Factor IX. Both types of hemophilia are associated with bleeding diatheses that can lead to crippling injuries or death. Traditionally, patients with either type of hemophilia were treated with infusion of plasma concentrates to stop bleeding episodes. The problems with the concentrates are many, especially infection, most recently with HIV. Highly purified Factor VIII or Factor IX have been introduced to obviate these problems, as have methods of treating factor concentrates to inactivate viruses. However, some patients develop high-titer, inhibitory antibodies to Factor VIII. Therefore, such patients can no longer be treated with conventional Factor VIII replacement therapy.
As described by Hedner and Kisiel, J Clin Invest 71:1836-1841 (1983), purified naturally produced Factor VIIa can be administered to hemophilia A patients with high titers of antibodies against Factor VIII:C and restore hemostasis in these patients. As reported by Brinkhous, et al., Proc Natl Acad Sci USA 86:1382-1386 (1989), recombinant Factor VIIa (rFVIIa) can be administered to hemophilic and von Willebrand disease dogs and stop bleeding in both hemophilic A and B dogs, but not the von Willebrand disease dogs. Telgt, et al., Thrombosis Res 56:603-609 (1989), reported that, at high levels, rFVIIa was believed to act by direct activation of Factor X, in the presence of calcium and phospholipid but in the absence of TF. Teitel, Thrombosis and Haemostasis 66:559-564 (1991), reported that the important ingredient in prothrombin complex concentrates for efficacy in treating hemophilia is Factor VIIa.
Hedner, "Experiences with Recombinant Factor VIIa in Haemophiliacs" in Biotechnology of Plasma Proteins. Curr Stud Hematol Blood Transf, Lenfant, Mannucci, Sixma, eds., No. 58, 63-68 (1991), review the use of prothrombin complex concentrates (effective in only 50 to 60% of the bleeds), as well as the use of plasma-derived (pFVIIa) and recombinant Factor VIIa (rFVIIa). Dosages of 10 to 15 .mu.g/kg of pFVIIa were effective in some hemophilia A patients. Safety studies in dogs and rabbits indicated that the recombinant Factor VIIa was safe and efficacious at dosages of up to 150 .mu.g/kg. A number of patients were also successfully treated, using dosages of between 54 .mu.g/kg and 90 .mu.g/kg during surgery and bleeding complications. Gringeri, et al., reported that treatment of hemophiliacs with rFVIIa is not always effective, even at dosages of 75 .mu.g/kg at intervals of every two to three hours. The authors noted that perhaps larger dosages, more frequent infusions, and/or the concomitant use of antifibrinolytic medication might be required in such cases. rFVIIa is currently in clinical trials in the United States for treatment of hemophilia, particularly hemophilia in patients with inhibitors who do not benefit from conventional Factor VIII or Factor IX replacement therapy. Doses of rFVIIa currently employed are typically 45 to 90 .mu.g rFVIIa/kg body weight, and are repeated every two to four hours. These doses are designed to achieve a level of circulating rFVIIa of approximately 4 .mu.g/ml, extremely high compared to the normal plasma concentrations of FVII of approximately 0.5 .mu.g/ml or FVIIa of approximately 1 ng/ml.
O'Brien, et al., J Clin Invest 82:206-211 (1988), reported that apo-TF, a delipidated preparation of the normally lipid-associated TF glycoprotein could be used to normalize bleeding in animals having antibodies to Factor VIII. Since purified apo-TF is inactive unless incorporated into a phospholipid membrane, the theoretical basis for infusing apo-TF is the hypothesis that it would spontaneously and preferentially incorporate into exposed membrane surfaces, particularly into damaged cells at the sites of injury. Subsequent studies have indicated there are dangers associated with the use of purified apo-TF in the treatment of hemophilia. The apo-TF can spontaneously incorporate into many types of lipid membranes and become active at sites where clotting is not desired, resulting in thrombosis or disseminated intravascular coagulation (DIC). Indeed, O'Brien, et al., reported evidence of DIC in some animals receiving apo-TF, and Sakai and Kisiel, Thromb Res 60:213-222 (1990), recently demonstrated that apo-TF will spontaneously combine with plasma lipoproteins to form active TF. This latter phenomenon is cause for concern because of a number of studies which have demonstrated that intravenous administration of active TF is a potent inducer of DIC.
Recently, a soluble, truncated form of TF (tTF) has been reported which retains some cofactor function towards Factor VIIa as measured in vitro using purified proteins. However, this form of TF has been dismissed as an alternative to TF because it has almost no procoagulant activity when tested with normal plasma, as reported by Paborsky, et al., J Biol Chem 266:21911-21916 (1991).
As described in U.S. Ser. No. 07/683,682 entitled "Quantitative Clotting Assay for Activated Factor VII" filed Apr. 10, 1991 by James H. Morrissey, the reason tTF was reported to lack procoagulant activity in the prior art is because, although tTF retains cofactor function toward Factor VIIa, tTF had lost the ability to promote conversion of Factor VII to Factor VIIa. As a consequence, tTF can clot plasma only in conjunction with significantly elevated levels of Factor VIIa, as compared to normal plasma, which contains only trace levels of Factor VIIa.
It is therefore an object of the present invention to provide a method and composition for treatment of significant bleeding disorders, such as hemophilia, including those hemophiliacs with high titers of anti-Factor VIII antibodies.
It is a further object of the present invention to provide a method and compositions for treatment of patients with bleeding disorders that are relatively safe and can be produced in commercial quantities.